Human immunodeficiency virus (HIV) infection rates and acquired immunodeficiency syndrome (AIDS) related death have reached pandemic proportions. According to the World Health Organization (WHO) and the joint United Nations Program on HIV/AIDS (UNAIDS), as of 2004, there were 39.4 million HIV infected adults and children, 4.9 million new infections (13,425 new infections every day), and 3.1 million AIDS related deaths had occurred worldwide. Recent projections from the WHO and UNAIDS, indicate that if the pandemic proceeds at its current rate there will be 45 million new HIV infections and 70 million deaths by 2020 (Stover et al., Lancet 2002 360:73-77).
Since its introduction in 1996, highly active antiretroviral therapy (3-drug combination therapy; “HAART”) has significantly reduced HIV-associated morbidity and mortality. HAART is seen to have successfully suppressed viral replication long-term, facilitated partial immune restoration, and prolonged survival. However, the incidence of HAART induced drug toxicity and the emergence of drug resistance has increased every year since its introduction. In addition, HAART regimens are expensive, have complex dose schedules and have significant drug-drug interactions. Accordingly, a novel therapeutic intervention that could complement HAART, shorten time on HAART, or even replace HAART in some HIV-infected subjects, would be a significant addition the anti-HIV armamentarium.
Given the inadequacy of HAART, there is a need for new treatment options. An immune-based therapy that can boost an HIV-infected subject's immune response and specifically enhance a CTL responses against HIV-1 has been proposed as possible strategy to limit the use and/or need for anti-retroviral medication (Kinloch-de Loes (2004) J. Antimicrob. Chemother. 53:562-566). Although several cell-based immunotherapeutics have been developed using consensus sequences of the HIV-1 viral genome as immunogens in viral vectors, the results of these clinical trials have been disappointing in their ability to suppress viral replication.
HIV immunotherapies based on clade-specific consensus antigens have been investigated in over 80 clinical trials, however, the results demonstrate a consistent lack of efficacy (Garber et al. (2004) The Lancet 4:397-413; McMichael (2006) Annual Rev. Immunol 24:227-255; and Nabel (2001) Nature 410:1002-1007). While augmentation of immune responses to consensus sequences used for immunization was demonstrated, these therapies did not result in reduction of viral loads. Evidence suggests that the lack of HIV-protective immunity is due to sequence divergence between autologous and consensus antigens. Studies with overlapping peptides demonstrated that CTL recognizing autologous peptides encoded within a known HIV virus did not cross react with corresponding consensus sequences (Altfeld et al. (2001) J. Exp. Med. 193:169-180). In addition, HIV's high mutation rate results in novel mutant variants that encode point mutations within CTL epitopes and escape recognition by specific T cells. Studies on humans and non-human primates correlate virus escape from CTL with progression to AIDS (Goulder et al. (1999) Mature Medicine 5:1233-1235; Goulder et al. (2004) Nature 4:630-640; and Barouch et al. (2002) Nature 415:335-339). In addition, each patient creates a unique environment for its own viral evolution. Consequently, there is substantial mutational variation between the virus infecting the patient and the reference sequences upon which most HIV immunotherapies are based. Since virus sequence diversity defines HIV clades, therapies based on consensus antigens from one clade may have limited ability to cross control evolutionally divergent viruses from other clades. Accordingly, therapies based on autologous viral antigens would have broader applicability since the therapy would be perfectly matched to the virus species infecting each subject.
To date, the successful dendritic cell (DC)-based immunotherapies for HIV infected patients used an autologous virus as a source of immunogen. One clinical study demonstrated that an AT-inactivated whole autologous HIV virus particle-loaded autologous DC therapy prolonged suppression of viral load by more than 90% in 8 patients for at least one year (Lu et al. (2004) Nature Medicine 10:1359-13565). All 8 of the enrolled subjects also maintained CD4+ T-cell counts. More recently, a second independent clinical study confirmed the benefit of immunizing patients against autologous virus (Garcia et al. (2005) J.I.D. 191:1680-1685). Patients in the later study were immunized with DC pulsed with autologous plasma-derived heat-inactivated virus. After immunization and interruption of HAART, set-point plasma viral load decreased by at least 0.5 log(10) copies/mL relative to baseline in 4 of 12 patients. This response was associated with changes in HIV-1-specific CD4+ lymphoproliferative and CD8+ T cell responses. Although these clinical studies demonstrated the potential utility of an autologous DC therapy, the choice of whole inactivated HIV virus as an immunogen is not ideal and may have significant safety and practical limitations.
Commonly owned PCT Publication WO2006/031870, the contents of which are incorporated by reference, discloses a method for strain-independent RT-PCR amplification of selected HIV antigens (such as Gag, Rev, Nef and Vpr) to generate templates for in vitro transcribed (IVT) RNA. The IVT RNA can be used to transfect dendritic cells or other antigen presenting cells (APCs) to produce an autologous RNA-loaded APC based HIV therapy with far lower regulatory hurdles (as the final formulation lacks infectious virus) in comparison to inactivated virus or total RNA vaccines.
Due to the high mutation rate of HIV, a major obstacle to RT-PCR amplification of autologous viral sequences was the design of PCR primers capable of amplifying variant HIV strains present in an individual without prior knowledge of the variant target sequences. PCT Publication WO2006/031870 solves this problem by disclosing a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient without the requirement of custom designing the primers for each of the variant viral sequences present in a particular individual. This approach to amplify HIV sequences in a clade-independent manner rests on the principles of multiplex RT-PCR technology. Pools of forward and reverse primers for each target gene (e.g., Gag, Rev, Vpr and Nef) are utilized such that most HIV strains will react with at least one forward and one reverse primer. Specifically, the amplification reactions use primer groups composed of primers that are complementary to a consensus target sequence as well as additional primers carrying compensatory mutations. A schematic diagram showing an embodiment of the RT-RCR amplification and in vitro transcription of HIV sequences strategy disclosed in WO2006/031870, using Rev as an example, is shown in FIG. 1. In FIG. 1, a Rev cDNA is made by reverse transcription using Rev specific primer group R8300, which contains multiple primer sequences. (Alternatively, the cDNA could be made using random primers to initiate reverse transcription.) The Rev cDNA is then amplified using three primary PCR reactions. Each primary PCR in this example utilizes the Rev R8300 reverse primer group, and one of three Rev forward primer groups (Rev F7750, Rev F7830, or Rev F7911). During the annealing step, the most complementary primer-template combination gives rise to a primary cDNA product. The primary cDNA is then amplified further in a secondary PCR reaction using groups of nested primers (the Rev T7 primer group and the Rev 64T primer groups are shown). Each 5′ nested primer contains a promoter (e.g., a T7 promoter) for subsequent in vitro transcription of the resulting cDNA product, while each 3′ nested primer preferably contains a 3′ polyT tract that can be transcribed into a polyA tail. The HIV cDNA produced by this multiplex RT-PCR-mediated amplification of autologous RNA encoding HIV antigens from small volumes of infectious plasma encode a complex mixture representing multiple quasispecies present within a given subject. The HIV cDNA produced by the secondary PCR can then be used for in vitro transcription to produce mRNA encoding the variant viral antigens.
DCs transfected with this IVT mRNA processed and presented multiple autologous HIV antigens and induce antigen-specific CD8+ T cells. The simultaneous use of autologous viral antigens and DCs provide for a broad patient-specific immune response that could potentially have a better control of residual virus or a rebound of virus following the cessation of therapy. This strategy can induce broad immune responses and the simultaneous assault on all relevant epitopes may have the added benefit of driving the virus into a state of poor replicative fitness and potentially eventual clearance. In addition, using specific RNAs to encode the antigens of interest instead of whole virus circumvents safety concerns. Furthermore, preliminary results from a Phase I/II clinical trial have demonstrated the safety of this approach in HIV patients.
Notwithstanding the success of the HIV amplification strategy disclosed in PCT Publication WO2006/031870, there is a need for primers that result in improved amplification of samples containing multiple quasispecies of HIV non-Clade B or Clade B nucleotides. In addition, it would be useful to develop primers that increase the yield of PCR products and/or in vitro transcripts. The present invention addresses these needs and provides additional advantages as well.